Traditional methods to detect DNA in a sample using polymerase chain reaction (PCR) detect double-stranded DNA, e.g., SYBR (Higuchi, U.S. Pat. No. 5,994,056) or use probe based detection. Examples of the latter method include TAQMAN™ (Gelfand et al., U.S. Pat. Nos. 5,210,015 and 5,487,972 and Livak et al., U.S. Pat. No. 5,723,591) and molecular beacons (Tyagi et al., International Publication No. WO 1995/013399). In TAQMAN™, a probe molecule that includes a fluorescent label and a quencher hybridizes to a PCR amplification product and is digested by the 5′ exonuclease activity of a polymerase. The 5′ exonuclease activity releases the fluorescent label from the probe, thereby separating the fluorescent label from the quencher and permitting detection of the fluorescent signal. One application of TAQMAN™ detection that finds use in SNP genotyping is described by Livak et. al (U.S. Pat. No. 5,538,848). The molecular beacons method utilizes a probe molecule having a stem-loop structure that keeps a fluorescent label and a quencher in close proximity. The probe molecule opens up upon binding to its complementary target, decreasing the quenching so that the fluorophore emits more light proportional to the number of target molecules amplified at a given cycle.
Whitcombe et al. describe the “Scorpion” method for DNA detection (U.S. Pat. No. 6,326,145 and 2005/0164219). This method employs a PCR primer with a 5′ tail that has a dye and a quencher. The 3′ end of the primer matches the target DNA in the sample, but the 5′ tail is complementary to the target amplicon this primer generates upon extension. Thus, the primer extension product forms a hairpin during PCR resembling a scorpion, and a fluorescent signal can be detected using the 5′ nuclease (e.g., TAQMAN™), molecular beacon or other method. Whitcombe et al. (Nature Biotechnology (1999) 17:804-807) describe unimolecular scorpion detection using “sunrise” primers that form a hairpin that brings a dye and a quencher close to each other; after primer extension, another hairpin forms with the dsDNA stem that increases the distance between the dye and the quencher. Thelwell et al. (Nucleic Acid Research (2000) 28: 3752-61) compared scorpion “molecular beacon” and 5′ nuclease detection with TAQMAN™ and showed that unimolecular scorpions give stronger signal than bimolecular TAQMAN™. Solinas et al. (Nucleic Acids Research (2001) 29(2):e96) compared molecular beacon and duplex scorpion with the quencher on a separate oligo and found that the latter provided larger signal difference between the quenched and unquenched states. The advantage of the scorpion method is that there is no separate probe molecule required to anneal to the amplicon (bimolecular interaction) and intramolecular hairpin formation has kinetic and thermodynamic (using the same probe versus hairpin stem sequence) advantages over intermolecular interactions. Using the scorpion method, hairpin formation will nearly always precede PCR primer annealing, whereas primer annealing often precedes probe to template annealing, leading to a decrease in detectable signal. For a review of various approaches for generating a detectable signal during quantitative PCR, as well as Cycling Probe Technologies (CPT)-based FEN, invader-type signal generation, and 5′ nuclease FRET signal, see Kutyavin, International Publication No. WO 2007/127999.
Methods that utilize universal primers and separate probes for nucleic acid detection have been described. For example, see Whitcombe et al. (U.S. Pat. No. 6,270,967) and Anderson et al. (U.S. Pat. No. 7,601,821). A drawback of these methods is that they are prone to generate non-specific signal. In these methods, a fluorescent signal is generated even if a single primer is involved in amplification. These methods generally use a multiplex pre-amplification (encoding), so inevitably primer dimers and non-specific amplifications occur. Even after the dilution that usually follows the pre-amplification step, these non-target amplicons and unused pre-amplification primers are still present in the mixture. Single primer specificity makes it more difficult to develop applications with “clean NTC” (“non template control”) signal.
The drawbacks of previously described detection methods that utilize target-specific primers and probes are cost and logistics. Scorpion approaches require a specific dual-labeled primer for each target nucleic acid (e.g., see U.S. Pat. No. 6,326,145), making these approaches quite expensive, especially when detection of multiple targets is required. For example, there are approximately 25,000 human genes and more than 10 million human single nucleotide polymorphisms (SNPs), and researchers often need to measure expression levels or determine the genotypes for tens, hundreds, or sometimes even thousands of targets. Furthermore, each user or research team/project generally requires a different set of genes/SNPs.
Several dyes are currently available as amidites, e.g., VIC and FAM. It is relatively inexpensive to manufacture oligonucleotides (“oligos”) with these dyes, as all of the oligo synthesis steps are performed on a column. There is a broad choice of fluorescent dyes that can be incorporated into oligos, but they require off-column dye attachment, e.g., CY3™, CY5™, and TEXASRED™. This makes oligo manufacturing more expensive, and often impractical, when each target requires a custom synthesis. Universal detection assays described hereinbelow amortize the oligo synthesis cost over multiple customers and experiments making it cost-effective to use the “off-column” dyes. Several commercial vendors preload assays in wells to simplify lab work for customers, e.g., TAQMAN™ low-density arrays and BIOTROVE™ from Life Technologies and SUPERARRAYS™ from Qiagen. Most customers require a custom set of assays, making manufacturing logistics for target-specific assays complicated. As described in detail herein, the present invention permits universal sets of assays for detecting any set of nucleic acid targets, making it much easier to offer preloaded assays: the same preloaded assays can be used to detect any set of targets. In addition, the present invention separates the encoding and detection steps, providing flexibility to detect multiple targets in the same color (in the same or different reaction volume), see, e.g., the examples for detecting trisomy 21 and HIV drug resistance mutations hereinbelow. Finally, unlike other universal detection methods, the present invention has two primer plus virtual probe specificity, rather than only two primer specificity for previously described universal detection formats.
Exiqon and Roche have developed the UNIVERSAL PROBELIBRARY™ (UPL) for gene expression that uses a set of 165 short 8-9 base universal probes (see Roche Applied Science website). These short probes have several locked nucleic acids (LNA, U.S. Pat. No. 6,670,461) that exhibit a high melting temperature when hybridized to DNA, in spite of being short. The universal probes also have normal DNA bases at the 5′ end so that they can be cleaved by the 5′-nuclease activity of a polymerase during PCR. The universal probes limit possible PCR primer locations in genes. However, the 165 universal probe set has sufficient occurrences in most mRNA sequences to enable choosing PCR primers such that at least one of the 165 universal sequences can be located between the two primers. The detection assays, however, are not universal, as each gene expression assay requires two target-specific primers.
Due to the above-noted drawbacks of current detection strategies (e.g., specificity, cost, manufacturing logistics, and the like), there is a need for more specific, flexible, and cost-effective methods of nucleic acid detection. The present invention meets these and a variety of other needs.